Team:Freiburg/Notebook/30 August

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Contents 1 green light receptor 2 PCR of green light promotor region 2.1 Ligation of Promotor PcpcG in PSB1C3 3 blue light receptor 3.1 Transformation 4 red light receptor 4.1 gel extraction 5 Lysis cassette 5.1 NAME OF YOUR EXPERIMENT 6 Precipitator

green light receptor

PCR

Name: Julia	Date: 8.08.2011
Project Name: PcpcG

 

PCR-Mixture for one Reaction:

For a 50 µl reaction use

32,5µl	H20
10µl	5x Phusion Buffer
2.5µl	Primer     up	tatgaattcgcggccgcttctagaCCATTGTGCTTTTCTCTATCAACC
2.5µl	Primer     dw	tatctgcagcggccgctactagtaACTTAAAAGTTGTTTAATGTCCAGCC
1µl	dNTPs
1µl	DNA-Template	Synechocystis genome
0.5 µl	Phusion (add in the end)

 

What program do you use?

 Temperature Time ( min)

94 C	5:00
94 C	00:30	30x
56 C	00:30	PCR of green light promotor region
72 C	1:00
72 C	7:00

To confirm the PCR-Product has the correct size, load 2 µl of the sample onto an agarose-gel.

 Size of expected gene : 238 bp

Ligation of Promotor PcpcG in PSB1C3

Investigators:Julia

1. Digestion of PCR Product from yesterday[1]

38µl PCR product
5µl BSA (10x)
5µl NEB buffer 4
1µl EcoRI
1µlPstI

Incubation for 3 hours, afterwards the sample was cleaned with a PCR purification kit from Qiagen.

2.Ligation

11µl H2O
4µl PcpcG
2µl pSB1C3
2µl T4 Ligase buffer
1µl T4 Ligase

Incubation for 3 hours at room temperature.
heat inactivation at 80 degrees for 20 min.

3. transformation

blue light receptor

Transformation

...with ♥-A3-Not, ♥-A3-nOt and ♥-A3-noT </br>

Investigators: Sophie

red light receptor

gel extraction

Investigators:Julia

Lysis cassette

NAME OF YOUR EXPERIMENT

Investigators:NAME

Precipitator

Cloning scheduleDate: 30.08.

Name: Rüdiger

1,2,3 : digest: Dpn1+BamH1+EcoR1

>PCR purification

>ligate into David Vector

8:digest:Dpn19EcoR1+psT1

>PCR purification

>ligate into CM Vector (Julia 25.08.)

4+9:PCR purification

>Gibson-Assembly (see SoP)

>PCR with P47+P44/45/46

>digest with Dpn1+EcoR1+PsT1

>ligate into PET-DUET1 Vector

>digest with Dpn1+EcoR1+Spe1

>ligate into CM vector

All PCR at 37°c for 2 hours without buffer.

Add all vectors to digest with Antarctic Phosphatase + AP buffer (5x).

Name Rüdiger	Date: 30.08.2011
Continue from Experiment PCR (Date) 29.08. (Name) Rüdiger
Project Name: Blue light receptor: Ligation of Lovtap and Not-Gate

Gibson-Assembly

1. Prepare 5X ISO buffer. Six ml of this buffer can be prepared by combining the following:

3 ml of 1 M Tris-HCl pH 7.5150 μl of 2 M MgCl₂60 μl of 100 mM dGTP 60 μl of 100 mM dATP 60 μl of 100 mM dTTP60 μl of 100 mM dCTP300 μl of 1 M DTT 1.5 g PEG-8000300 μl of 100 mM NADAdd water to 6 ml Aliquot 100 μl and store at -20 °C

2. Prepare an assembly master mixture. This can be prepared by combining the following:

320 μl 5X ISO buffer0.64 μl of 10 U/ μl T5 exo20 μl of 2 U/μl Phusion pol160 μl of 40 U/μl Taq ligAdd water to 1.2 ml

Aliquot 15 μl and store at -20 °C. This assembly mixture can be stored at -20 °C for at least one year. The enzymes remain active following at least 10 freeze-thaw cycles. This is ideal for the assembly of DNA molecules with 20-150 bp overlaps. For DNA molecules overlapping by larger than 150 bp, prepare the assembly mixture by using 3.2 μl of 10 U/ μl T5 exo.

3. Thaw a 15 μl assembly mixture aliquot and keep on ice until ready to be used.

4. Add 5 μl of DNA to be assembled to the master mixture. The DNA should be in equimolar amounts. Use 10-100 ng of each ~6 kb DNA fragment. For larger DNA segments, increasingly proportionate amounts of DNA should be added (e.g. 250 ng of each 150 kb DNA segment).

5. Incubate at 50 °C for 15 to 60 min (60 min is optimal).

6. If cloning is desired, electroporate 1 μl of the assembly reaction into 30 μl electrocompetent E. coli.

Documentation:

Why are you doing this experiment? Name the parts for the Gibson-Assembly.

Precipitator 2 : 4+9 42: 5+10 4:6+11

Describe your results and mistakes. Did you digest it? Results?

See gel next day >Did not work due to wrong DNA concentrations and no PCR purification

How did you label your samples and where are they stored?

Digestion this time the vector is dephosphorilated by antarctic phosphatase to avoid religation.

Name: Sophie	Date: 31.08.11
Continue from Date: 30.08.11 Name: Sophie Experiment: new
Project Name:inducible promoter for pbd (iGEM standard)

Procedure

1. add H₂O (38μl-DNA )
2. 5 μl NEB4 buffer (stored at iGEM’s, -20°C)
3. 5 μl 10x BSA (used 1:10 diluted sample stored at iGEM’s, -20°C)
4. DNA (500 ng) (Vector:Insert ratio 1:3 in following Ligation)
5. 1 μl restriction enzymes (stored at iGEM’s, -20°C)
6. heat for 1-2 hours 37°C (6 hours if time)
7. add 5,6 μl antarctic phosphatase buffer and 1 μl antarctic phosphatase and incubate for another hour
8. heat for 20 minutes 80°C (inactivation of enzymes)
9. keep at 4°C if you cannot continue

Measured DNA-concentration with Nanodrop to calculate the volume of DNA to do the digestion:

Sample Name	DNA concentration (μg/μl)
GFP-pbd	164
IPTG-promoter ε 5	217
psB1C3	58

Restriction enzymes you need to cut the vector, insert1 and insert 2:

Components	Vector (μl)		Insert1 and 2 (μl)
DNA (500ng)
BSA (10x) (5μl)
NEB4 Buffer (5μl)
Enzyme 1 (1μl)		Eco		Eco	Xba
Enzyme 2 (1μl)		Pst		Spe	Pst
H2O (38 μl- DNA)
In total 50 μl

Documentation:

Why are you doing this experiment? Where are the samples stored? Antibiotica resistance, vector used etc.

I want to be able to send the plastic binding domain to the registry and I want it to be expressible. Stored in: “Minipreps, verdaut”-box Resistance: Cm